1. Field of the Invention
The present invention relates to conductive carbon nanotubes (CNTs) obtained by dotting carboxylated carbon nanotubes with metal nanocrystals by chemical functional groups. Also, the invention relates to a method for fabricating a conductive CNT pattern or film, which comprises repeatedly depositing such conductive CNTs on a substrate to have high surface density. Moreover, the invention relates to a biosensor wherein bioreceptors that bind to target biomolecules are selectively attached to the conductive CNTs or the conductive CNT pattern or film, as well as to a fabricating method for such biosensor.
2. Background of the Related Art
Carbon nanotube (CNT) is an allotrope of carbon, which is composed of carbons that exist abundantly on the earth. CNTs are tubular materials in which a carbon atom is connected to other carbons in the form of a hexagonal honeycomb structure. Their diameter is about the size of nanometer ( 1/109 meter). CNT is known to have excellent mechanical properties, electrical selectivity, field emission properties and highly efficient hydrogen storage properties and is substantially defect-free in character.
CNT in consequence of such attributes has virtually limitless applicability in the fields of electron emitters, vacuum fluorescent displays (VFD), white luminous sources, field emission displays (FED), lithium ion secondary battery electrodes, hydrogen storage fuel cells, nano-wires, nano-capsules, nano-tweezers, AFM/STM tips, single electron devices, gas sensors, medical engineering microscopic parts, etc.
Because of their properties of excellent structural rigidity, chemical stability, and ability to act as ideal one-dimensional (1D) “quantum wires” with either semiconducting or metallic behaviors and a large aspect ratio, CNT exhibits a broad range of potential applications as a basic material of flat panel displays, transistors, energy reservoirs, etc., and as various sensors with nanosize (Dai, H., Acc. Chem. Res., 35:1035, 2002).
In order to apply such properties more diversely, the purified single-walled CNT has been cut into short nanotube pieces using an acid. The cut CNT pieces have mainly —COOH chemical functional groups at a part of ends and sidewall of the open tube. The properties of the CNT have been modified by chemical binding of various materials using these chemical functional groups. Further, substitution of the functional group of CNT for an —SH group by chemical manipulation and patterning on a gold surface using a microcontact printing method has been reported (Nan, X. et al., J. Colloid Interface Sci., 245:311, 2002). CNT immobilization on a substrate in a multilayered film using an electrostatic method has also been reported (Rouse, J. H. et al., Nano Lett., 3:59, 2003). The first-mentioned substitution method has disadvantages of low CNT surface density and weak bonding, and the second-mentioned CNT immobilization method also has the fatal disadvantage that the patterning method for selective immobilization on the surface cannot be applied. Therefore, there is an urgent need for a new type of surface immobilizing method that achieves high density.
Since most diseases are caused at a protein level other than a genetic level, more than 95% of medical drugs developed to date or in current development, target a protein. Thus, technologies for the detection of protein-protein and protein-ligand interactions are necessary in studies to establish the function of biomolecules interacting with certain proteins and ligands and to develop therapeutic and preventive methods against diseases. The development of such technologies by classical techniques, based on data obtained by protein function analysis and network analysis, has not been successful in providing a simple, economic, effective and reliable method for detection of protein-protein and protein-ligand interactions.
The technology for the detection of protein-protein interaction, as heretofore practiced, is a protein-chip technology. This is a technology in which the orientation of biomolecules is controlled at a molecular level using an affinity tag for a target protein, to specifically immobilize a uniform stable monolayer of protein on the surface of a substrate, followed by the analysis of the protein-protein interaction (Hergenrother, P. J. et al., JACS, 122:7849, 2000; Vijayendran, R. J., A. et al., Anal. Chem., 73:471, 2001; Benjamin, T. et al., Tibtech., 20:279, 2002).
Recently, research has been directed to the detection of both protein-protein and protein-ligand reactions by means of electrochemical changes of CNT after immobilization of a biomaterial (Dai, H. et al., ACC. Chem. Res., 35:1035, 2002; Sotiropoulou, S. et al., Anal. Bioanal. Chem., 375:103, 2003; Erlanger, B. F. et al., Nano Lett., 1:465, 2001; Azamian, B. R. et al., JACS, 124:12664, 2002). A representative example of a protein-ligand reaction is an avidin-biotin reaction. In one reported effort, a channel was formed on a substrate that had been treated with a polymer, using CNT and the binding activity of streptavidin was measured by means of an electrochemical method (Star, A. et al., Nano Lett., 3:459, 2003).
The methods of preparing a high density CNT structure, attaching DNA thereon and detecting complementary DNA are useful in genotyping, mutation detection, pathogen identification and the like. It has been reported that PNA (peptide nucleic acid: DNA mimic) is regio-specifically fixed on a single walled CNT and complementary binding to probe DNA is detected (Williams, K. A. et al., Nature, 420:761, 2001). The fixture of an oligonucleotide on a CNT array by an electrochemical method and detection of DNA by guanidine oxidation also has been reported (Li, J. et al., Nano Lett., 3:597, 2003). However, these methods do not apply CNT to fabrication and development of biochips.
Recently, a high capacity biomolecule detection sensor using CNT has been disclosed (WO 03/016901 A1). This patent publication relates to a multi-channel type biochip produced by arranging a plurality of CNTs on a substrate using a chemical linker and attaching various types of receptors. However, it has the disadvantage that precision analysis is not achieved, due to the relative weakness of electric conductivity of the sensor.
The reasons that CNT attracts public attention as a biochip material include the following: firstly, CNT needs no labeling; secondly, CNT has high sensitivity to signal change; and thirdly, CNT is capable of reacting in an aqueous solution without deterioration of a protein. The combination of a new nanomaterial and a biological system will create important fusion technologies in a large number of fields, including disease diagnosis (hereditary diseases), proteomics and nanobiotechnology.
A large amount of genetic information was obtained by the Human Genome Project, and this information has provided a stepping-stone that will lead to innovation in the understanding and diagnosis of genetic diseases. In this effort, the development of an effective DNA fingerprinting system for genomic sequencing, mutation detection and pathogen identification is needed.
In order to develop a faster and cheaper biosensor, substantial research efforts have been focused on technologies of DNA hybridization detection. Various labeling techniques for detecting DNA hybridization have been developed. Currently, fluorescent substances are most generally used in labeling. A single DNA chain capable of detecting complementary DNAs is immobilized to recognize complementary DNAs in aqueous solution, and a signal transducer changes a DNA hybridization signal into an analyzable signal.
Regarding the signal transducers, optical (fluorescent), piezoelectric and electrochemical transduction techniques are being studied. Among these, the electrochemical technique has various advantages, including high sensitivity, low cost and compatibility with microfabrication technology, and it can detect DNA having specific base sequences in a rapid and direct manner.
There are several methods capable of immobilizing a DNA probe on a transducer surface. These methods can be classified into several categories, including chemical adsorption, covalent-binding, electrostatic attraction, co-polymerization, and avidin-biotin affinity approaches. Also, DNAs may be immobilized on a micrometer-sized surface using a conductive polymer.
An effective surface treatment capable of increasing hybridization efficiency and simultaneously, removing the background from non-specific binding, is required to detect the DNA hybridization effectively using a DNA chip. Much research has been conducted to prepare a surface-treated DNA chip platform (Rogers, Y. et al., Anal. Biochem., 266:23, 1999; Hu, J. et al., Nuc. Acid. Res., 29:106, 2001). Also, various methods for detecting DNA hybridization have been developed, which include the scanometric method, the calorimetric method, a nanoparticle method, an electrochemical method, and etc. (Taton, T. A. et al., Science, 289:1757, 2000; Alexandre, I. et al., Anal. Biochem., 295:1, 2001; Cai, H. et al., Analyst., 127:803, 2002; Cai, H. et al., Anal. Bioanal. Chem., 375:287, 2003).
Additionally, many applications of CNT in the bioengineering field have recently begun appearing in the literature, including application of CNT to biochips, such as glucose biosensors, detecting protein, detecting a certain DNA sequence, and the like (Sotiropoulou, S. et al., Anal. Bioanal. Chem., 375:103, 2003; Chen, R. J. et al., Proc. Natl. Acad. Sci. USA, 100:4984, 2003; Cai, H. et al., Anal. Bioanal. Chem., 375:287, 2003). Screening bio-molecules from a multilayer based on CNT can increase the amount of immobilized bio-substances, such as DNAs, and can increase the detection sensitivity to the bio-substances, since multilayer structures based on CNT have wide surface area and high electric conductivity.
The recent efforts to combine biotechnology (BT) with nanotechnology (NT) has accelerated the development of hybrid nanomaterials using the property of biomaterials that can specifically bind. DNAs are of particular interest as smart nanowires that can bind to the desired locations.
The combination of information technology (IT), NT and BT has made it possible to employ quick and precise digital information in the measurement of analog data, such as the presence or absence of biomaterials, and reactivity, by electrical detection methods (Chen, J. et al., JACS, 122:657, 2000; Dahne, L. et al., JACS, 123:5431, 2001).
A lipid-protein double layer which was first examined has electrical properties. Thus, it was used in cell immobilization to study cell surface characteristics and cell interactions. A more practical application has used a receptor layer as a biosensor for optical and electrical detection. In 1993, German Stelzle, M. et al. reported its possibility as a biosensor by impedance analysis in a sensor based on two layers of lipid/receptor (Stelzle, M. et al., J. Phys. Chem., 97:2974, 1993). Furthermore, in the field of detecting smaller molecules by electrical methods, a study result was reported indicating that the directions of electric dipoles in organic molecules can be controlled in nanoscale by applying electric pulses to a probe, such as an atomic force microscope (AFM). According to this study, if the probe is coated with a suitable metal to confer electrical properties, and electric pulses with changed polarity are applied to the organic molecules from the probe, high-density molecular memory chips, such as devices, can be fabricated, and electric charge of organic molecules can also be measured by this approach (Matsushige, K. et al., Nanotechnol., 9:208, 1998).
An international patent application relating to methods of comparing the relative contents of biomolecules and identifying biomolecules in a sample with affinity tags and mass spectrometry was recently published (WO 2002/86168 A1).
At present, the most universal method for detecting the result of a reaction in a biochip is to use conventional fluorescent materials and isotopes (Toriba, A. et al., Biomed. Chromatogr., 17:126, 2003; Syrzycka, M. et al., Anal. Chim. Acta, 484:1, 2003; Grow, A. E. et al., J. Microbio. Meth., 53:221, 2003). However, as novel methods to more readily and precisely measure an electrical or electrochemical signal develop, there are increased demands for CNT as a new material.